Biology subjects

Steven, B.

Publications and source records attributed to Steven, B..

13 recordsLinked to original sources

bioRxiv2026

A qPCR method facilitates study of absolute abundance, ecology, and inoculation fate of ciliate predators on the leaf surface

Predatory protists are important in shaping terrestrial microbial ecosystems, but their roles in the phyllosphere, or the communities on aerial plant surfaces, are poorly understood. Previous work found that the order Colpodida dominated heterotrophic protist communities in the phyllosphere. While most protists were sporadically present, a few Colpodida variants were prevalent and abundant, indicating that these variants may represent species adapted to the phyllosphere. To identify these organisms, we cultured colpodids from field-collected tomato leaves and performed phylogenetic analysis of the 18S rRNA gene. Five of nine independent isolates matched the most prevalent Colpodida variant previously identified as leaf-enriched through amplicon sequencing, and these isolates comprised a novel clade of Paracolpoda steinii. When compared to a maize root isolate of Colpoda inflata, an abundant rhizosphere ciliate, a P. steinii isolate was similar in size and growth yield on E. coli, but grew to higher yields and formed large cyst clusters when incubated with model phyllosphere bacteria prey Erwinia and Pseudomonas. We developed and validated quantitative PCR (qPCR) methods for detection and cell abundance estimation of the P. steinii phyllosphere clade, C. inflata, and the order Colpodida in environmental samples. In inoculated greenhouse plants, qPCR-estimated protist populations matched measured inoculum levels, and protist inoculum was still detectable after five days. In an uninoculated tomato field, P. steinii was detected on all plants, with greatest abundances observed in lower leaves and after a rain event. P. steinii comprised up to 18.7% of total leaf Colpodida populations, which were estimated at up to [~]1400 organisms per gram of fresh weight. The findings demonstrate that Colpodida communities are consistently present on tomato leaves, dynamically affected by the abiotic environment, and include significant populations of P. steinii. We propose that the P. steinii isolates and qPCR tools presented can be used as a model system to investigate colonization and distribution patterns, biotic interactions, genetic adaptations, and agricultural applications of leaf predation.

microbiology↗

bioRxiv2025

Rhizosphere-colonizing bacteria persist in the protist microbiome

Soils contain diverse predatory protists that affect the abundance and behavior of rhizosphere bacteria, including bacteria that may benefit plant health. Protists also harbor their own bacterial microbiomes, including transient and extracellular associates, but it is not known whether the protist microbiome affects the plant rhizosphere. To address this question, we profiled the bacterial microbiomes of eight evolutionarily diverse rhizosphere protist isolates after two years of continuous laboratory culture. We then compared the protist culture microbiomes to maize rhizosphere communities six weeks after protist inoculation. Introduction of protists enriched 13 protist-associated bacterial amplicon sequence variants (ASVs) in the rhizosphere, which comprised [~]10% of the rhizosphere bacterial community. Additional bacterial ASVs ranked highly in abundance in both rhizosphere and protist microbiomes; together, a median 47% of the protist microbiome was enriched or abundant in the rhizosphere. Inoculation with some of the protist cultures positively affected root biomass traits, but a protist mixture had no effect, indicating that the impact of protist holobionts on plant growth is context-dependent. Isolates of protist-associated bacteria had both positive and negative effects on protist growth in culture, suggesting that the bacteria use multiple strategies to survive in proximity to predators. This study demonstrates that even after prolonged laboratory culture, evolutionarily diverse rhizosphere protists host bacterial microbiomes dominated by plant-colonizing bacteria that impact the rhizosphere microbiome after inoculation. The findings suggest that protists may contribute to the rhizosphere as part of the soil microbial seedbank, and identify bacterial groups that may be important to the plant-protist interaction. ImportanceUnderstanding the impact of predatory protists on the plant microbiome will be essential to deploy protists in sustainable agriculture. This study shows that eight rhizosphere protist isolates hosted diverse and distinct bacterial communities, and that a large proportion of these bacteria could be found colonizing the maize root environment long after protists were introduced. This study demonstrates that maize rhizosphere bacteria can persist for years in the protist microbiome, indicating that extremely diverse eukaryotes could help select and maintain rhizosphere bacteria in the absence of the plant.

microbiology↗

bioRxiv2023

Establishment and comparative genomics of a high-quality collection of mosquito-associated bacterial isolates -- MosAIC (Mosquito-Associated Isolate Collection)

Mosquitoes transmit medically important human pathogens, including viruses like dengue virus and parasites such as Plasmodium spp., the causative agent of malaria. Mosquito microbiomes are critically important for the ability of mosquitoes to transmit disease-causing agents. However, while large collections of bacterial isolates and genomic data exist for vertebrate microbiomes, the vast majority of work in mosquitoes to date is based on 16S rRNA gene amplicon data that provides limited taxonomic resolution and no functional information. To address this gap and facilitate future studies using experimental microbiome manipulations, we generated a bacterial Mosquito-Associated Isolate Collection (MosAIC) consisting of 392 bacterial isolates with extensive metadata and high-quality draft genome assemblies that are publicly available for use by the scientific community. MosAIC encompasses 142 species spanning 29 bacterial families, with members of the Enterobacteriaceae comprising 40% of the collection. Phylogenomic analysis of three genera, Enterobacter, Serratia, and Elizabethkingia, reveal lineages of mosquito-associated bacteria isolated from different mosquito species in multiple laboratories. Investigation into species pangenomes further reveals clusters of genes specific to these lineages, which are of interest for future work to identify functions underlying mosquito host association. Altogether, we describe the generation of a physical collection of mosquito-associated bacterial isolates, their genomic data, and analyses of selected groups in context of genome data from closely related isolates, providing a unique, highly valuable resource to investigate factors for bacterial colonisation and adaptation within mosquito hosts. Future efforts will expand the collection to include broader geographic and host species representation, especially from individuals collected from field populations, as well as other mosquito-associated microbes, including fungi, archaea, and protozoa.

microbiology↗

bioRxiv2023

Impact of inter-species interactions between flower microbiota commensals and a floral pathogen on disease incidence and pathogen activity

Flowers are colonized by a diverse community of microorganisms that alter plant health and interact with floral pathogens. Erwinia amylovora is a flower-inhabiting bacterium and a pathogen that infects different plant species, including Malus x domestica (apple). Previously, we showed that the co-inoculation of two bacterial strains, Pseudomonas and Pantoea natural inhabitants of apple flowers, reduced disease incidence caused by this floral pathogen. Here, we decipher the ecological interactions between these two flower-associated bacteria and E. amylovora in field experimentation and in vitro co-cultures. The two flower microbiota strains did not competitively exclude E. amylovora from the stigma habitat, as both commensal bacteria and the pathogen co-existed on the apple flowers, proscribing microbial antibiosis or niche exclusion as predominant mechanisms of host protection. Inspection of the total and the active microbiota populations on flowers revealed that strain co-inoculations altered microbiota activity. Using synthetic stigma exudation medium, tripartite co-culture of the bacterial strains strongly acidified the growth milieu and led to a substantial alteration of gene expression in both the pathogen and the two microbiota members. Our study emphasizes the critical role of emergent properties mediated by inter-species interactions within the plant holobiont and their impact on plant health and pathogen behavior. ImportanceFire blight, caused by Erwinia amylovora, is one of the most important plant diseases of pome fruits. Plant-associated microbiota can influence plant disease occurrence through inter-species interactions. Previous studies have shown that plant microbiota commensals could suppress disease mainly by antagonizing of the pathogen growth, however, whether plant-associated microbiota could alter pathogen activity and behavior have not been well studied. Here, we show that the co-inoculation of two bacterial strains that naturally colonize the apple flowers reduce disease pressure. We further demonstrate that the interactions between these two microbiota commensals and the floral pathogen lead to a strong alteration of the pH and to the emergence of new gene expression patterns that may alter the pathogen behavior. Our findings emphasize the critical role of emergent properties mediated by inter-species interactions between plant microbiota and plant pathogens and their impact on plant health.

microbiology↗

bioRxiv2023

Three Species of Axenic Mosquito Larvae Recruit a Shared Core of Bacteria in a Common Garden Experiment

In this study we describe the generation of two new species of axenic mosquito, Aedes albopictus and Aedes triseriatus. Along with Aedes aegypti, axenic larvae of these three species were exposed to an environmental water source to document the assembly of the microbiome in a common garden experiment. Additionally, the larvae were reared either individually or combinatorially with the other species to characterize the effects of co-rearing on the composition of the microbiome. We found that the microbiome of the larvae was composed of a relatively low diversity collection of bacteria from the colonizing water. The abundance of bacteria in the water was a poor predictor for their abundance in the larvae, suggesting the larval microbiome is made up of a subset of relatively rare aquatic bacteria. We found eleven bacterial 16S rRNA gene amplicon sequence variants (ASVs) that were conserved amongst >90% of the mosquitoes sampled, including two found in 100% of the larvae, pointing to a conserved core of bacteria capable of colonizing all three species of mosquito. Yet, the abundance of these ASVs varied widely between larvae suggesting individuals harbored largely unique microbiome structures, even if they overlapped in membership. Finally, larvae reared in a tripartite mix of the host species consistently showed a convergence in the structure of their microbiome, indicating that multi-species interactions between hosts potentially lead to shifts in the composition of their respective microbiomes. IMPORTANCEThis study is the first report of the axenic (free of external microbes) rearing of two species of mosquito, Aedes albopictus and Aedes triseriatus. With our previous report of axenic Aedes aegypti, brings the number of axenic species to three. We designed a method to perform a common garden experiment to characterize the bacteria the three species of axenic larvae assemble from their surroundings. Furthermore, species could be reared in isolation or in multi-species combinations to assess how host species interactions influence the composition of the microbiome. We found all three species recruited a common core of bacteria from their rearing water, with a large contingent of rare and sporadically detected bacteria. Finally, we also show that co-rearing of mosquito larvae leads to a coalescence in the composition of their microbiome, indicating that host species interactions potentially influence the composition of the microbiome.

microbiology↗

bioRxiv2021

A dual sequence and culture-based survey of maize rhizosphere protists reveals dominant,plant-enriched, and culturable community members

Protists play important roles in shaping the microbial community of the rhizosphere. However, there is still a limited understanding of how plants shape the protist community, and how well protist isolate collections might represent rhizosphere protist composition and function in downstream studies. We sought to determine whether maize roots select for a distinct protist community in the field, and whether the common or dominant members of that community are readily culturable using standard protist isolation methods. We sequenced 18S and 16S rRNA genes from the rhizospheres of maize grown in two sites, and isolated 103 protists into culture from the same roots. While field site had the greatest effect, rhizospheres in both sites had distinct protist composition from the bulk soils, and certain taxa were enriched in both sites. Enriched taxa were correlated to bacterial abundance patterns. The isolated protists represented six supergroups, and the majority corresponded to taxa found in the sequencing survey. Twenty-six isolates matched eight of the 89 core rhizosphere taxa. This study demonstrates that maize roots select for a distinct protist community, but also illustrate the potential challenges in understanding the function of the dominant protist groups in the rhizosphere. Originality-Significance StatementThis is the first study comparing cultivation-dependent and independent methods for studying the protist community of plant roots, and the first untargeted analysis of the maize rhizospheres effect on protist communities. We show that maize in different sites select for distinct communities and overlapping enriched taxa, but that isolating the most important plant-associated protists may be a challenge for researchers.

microbiology↗

bioRxiv2021

Whats under the Christmas tree? Soil acidification alters fir tree rhizosphere bacterial and eukaryotic communities, their interactions, and functional traits

pH has been identified as a master regulator of the soil environment, controlling the solubility and availability of nutrients. As such, soil pH exerts a strong influence on indigenous microbial communities. In this study we describe a soil acidification experiment and the resulting effects on the rhizosphere communities of fir trees on a Christmas tree plantation. The acidification treatment reduced the pH of bulk soil by [~]1.4 pH units and was associated with reduced Ca, Mg, and organic matter content. Similarly, root chemistry differed due to soil acidification with roots in acidified soils showing significantly higher Al, Mn, and Zn content and reduced levels of B and Ca. 16S rRNA and 18S rRNA gene sequencing was pursued to characterize the bacterial/archaeal and eukaryotic communities in the rhizosphere soils. The acidification treatment induced dramatic and significant changes in the microbial populations, with thousands of 16S RNA gene sequence variants and hundreds of 18S rRNA gene variants being significantly different in relative abundance between the treatments. Additionally, co-occurrence networks showed that bacterial and eukaryotic interactions, network topology, and hub taxa were significantly different when constructed from the control and acidified soil rRNA gene amplicon libraries. Finally, metagenome sequencing showed that the taxonomic shifts in the community resulted in alterations to the functional traits of the dominant community members. Several biochemical pathways related to sulfur and nitrogen cycling distinguished the metagenomes generated from the control and acidified soils, demonstrating the myriad of effects soils acidification induces to rhizosphere microbes. IMPORTANCESoil pH has been identified as the property that exerts the largest influence on soil microbial populations. We employed a soil acidification experiment to investigate the effect of lowering soil pH on the bacterial and eukaryotic populations in the rhizosphere of Christmas trees. Acidification of the soils drove alterations of fir tree root chemistry and large shifts in the taxonomic and functional composition of the communities, involving pathways in sulfur and nitrogen cycling. These data demonstrate that soil pH influences are manifest across all organisms inhabiting the soil, from the host plant to the microorganisms inhabiting the rhizosphere soils. Thus, pH is an important factor that needs to be considered when investigating soil and plant health, the status of the soil microbiome, and terrestrial nutrient cycling.

microbiology↗

bioRxiv2020

Validation of a PNA clamping method for reducing host DNA amplification and increasing eukaryotic diversity in rhizosphere microbiome studies

Protists and microscopic animals are important but poorly understood determinants of plant health. Plant-associated eukaryotes could be surveyed by high-throughput sequencing of 18S ribosomal RNA (rRNA) genes, but the abundance of plant DNA in rhizosphere samples makes 18S rRNA gene amplification with universal primers unfeasible. Here we applied a pipeline to generate peptide nucleic acid (PNA) clamps to suppress the amplification of plant host DNA during 18S rRNA gene library preparation. We designed a PNA clamp, PoacV9_01, specific to the V9 region of the 18S rRNA gene for plants in the family Poaceae. PoacV9_01 suppressed the amplification of five species of grain crops in quantitative PCR reactions. In an 18S rRNA gene sequencing survey of the rhizosphere of maize, PoacV9_01 reduced the relative abundance of plant reads from 65% to 0.6%, while drastically increasing the number and diversity of animal, fungal, and protist reads detected. Thus, PoacV9_01 can be used to facilitate the study of eukaryotes present in grass phytobiomes. In addition, the pipeline developed here can be used to develop PNA clamps that target other plant species.

microbiology↗

bioRxiv2020

Temporal and spatial dynamics in the apple flower microbiome in the presence of the phytopathogen Erwinia amylovora

Plant microbiomes have important roles in plant health and productivity. However, despite flowers being directly linked to reproductive outcomes, little is known about the microbiomes of flowers and their potential interaction with pathogen infection. Here, we investigated the temporal dynamics and spatial traits of the apple stigma microbiome when challenged with a phytopathogen Erwinia amylovora, the causal agent of fire blight disease. We profiled the microbiome from the stigmas of a single flower, greatly increasing the resolution at which we can characterize shifts in the composition of the microbiome. Individual flowers harbored unique microbiomes at the OTU level. However, taxonomic analysis of community succession showed a population gradually dominated by bacteria within the families Enterobacteriaceae and Pseudomonadaceae. Flowers inoculated E. amylovora established large populations of the phytopathogen, with pathogen specific gene counts of >3.0 x 107 in 90% of the flowers. Yet, only 42% of inoculated flowers later developed fire blight symptoms. This reveals pathogen amount on the stigma is not sufficient to predict disease outcome. Our data demonstrate that apple flowers represent an excellent model in which to characterize how plant microbiomes establish, develop, and interact with biological processes such as disease progression in an experimentally tractable plant organ.

microbiology↗

bioRxiv2019

Antibiotic resistant bacteria and commensal fungi are common and conserved in the mosquito microbiome

The emerging and increasing prevalence of bacterial antibiotic resistance is a significant public health challenge. To begin to tackle this problem, it will be critical to not only understand the origins of this resistance but also document environmental reservoirs of antibiotic resistance. In this study we investigated the possibility that both colony and field caught mosquitoes could harbor antibiotic resistant bacteria. Specifically, we characterized the antibiotic resistant bacterial populations from colony-reared Aedes aegypti larvae and adults and two field caught mosquito species Coquillettidia perturbans and Ochlerotatus canadensis. The cultured bacterial populations were dominated by isolates belonging to the class Gammaproteobacteria. Among the antibiotic resistant populations, we found bacteria resistant to carbenicillin, kanamycin, and tetracycline, including bacteria resistant to a cocktail of all three antibiotics in combination. The antibiotic resistant bacteria were numerically rare, at most 5% of total cell counts. Isolates were characterized by 16S rRNA gene sequencing, and clustering into Operational Taxonomic Units (OTUs; 99% sequence identity). 27 antibiotic resistant OTUs were identified, although members of an OTU did not always share the same resistance profile. This suggests the clustering was either not sensitive enough to distinguish different bacteria taxa or different antibiotic resistant sub-populations exist within an OTU. Finally, the antibiotic selection opened up a niche to culture mosquito-associated fungi, and 10 fungal OTUs (28S rRNA gene sequencing) were identified. Two fungal OTUs both classified to the class Microbotryomycetes were commonly identified in the field-caught mosquitoes. Thus, in this study we demonstrate that antibiotic resistant bacteria and certain fungi are common and conserved mosquito microbiome members. These observations highlight the potential of invertebrates to serve as vehicles for the spread of antibiotic resistance throughout the environment.

microbiology↗

bioRxiv2018

Biogeography and Edaphic Factors Structure Coastal Sediment Microbial Communities More than Vegetation Removal by Sudden Vegetation Dieback

Development of sudden vegetation dieback (SVD), a phenomenon that causes the rapid mortality of salt marsh plants, specifically Spartina alterniflora, has affected large-scale alterations in Atlantic coastal systems, through the often-complete removal of vegetation. In this study, two wetlands that differ in the time since development of SVD were compared in order to study biogeographic and temporal patterns that structure coastal wetland microbial communities and their response to disturbance.\n\nBiogeographic and edaphic factors that distinguished the two wetlands, such as differing salinity, water content, and soil carbon and nitrogen between the sites were more strongly associated with sediment microbial community structure than either sampling date or SVD development. In fact, no OTUs differed in abundance due to the season samples were collected, or vegetation loss due to SVD. This is not to say that SVD did not alter the composition of the microbial communities. The taxonomic composition of sediment communities in SVD-affected sediments was more heterogeneous between samples and a small number of OTUs were enriched in the vegetated sediments. Yet, these data suggest that coastal wetland sediment communities are predominantly shaped by environmental conditions and are generally resilient to temporal cycles or ecosystem disturbances.\n\nImportanceOne of the challenges of microbial ecology is predicting how microbial communities will respond to ecosystem change. Yet, few studies have addressed whether microbial responses to disturbance are consistent over space or time. In this study we employ SVD as a natural vegetation removal experiment and compare the sediment microbial communities between two geographically separated wetlands (ca 125 km). In this manner, we uncover a hierarchical structuring of the microbial communities, being predominantly governed by biogeography, with lesser effects due to disturbance, or temporal dynamics.

microbiology↗

bioRxiv2018

Chronic Physical Disturbance Substantially Alters the Response of Biological Soil Crusts to a Wetting Pulse, as Characterized by Metatranscriptomic Sequencing

Biological soil crusts (biocrusts) are microbial communities that are a feature of arid surface soils worldwide. In drylands where precipitation is pulsed and ephemeral, the ability of biocrust microbiota to rapidly initiate metabolic activity is critical to their survival. Community gene expression was compared after a short duration (1 hour) wetting pulse in both intact and soils disturbed by chronic foot trampling.\n\nAcross the metatranscriptomes the majority of transcripts were cyanobacterial in origin, suggesting that cyanobacteria accounted for the bulk of the transcriptionally active cells. Chronic trampling substantially altered the functional profile of the metatranscriptomes, specifically resulting in a significant decrease in transcripts for nitrogen fixation. Soil depth (biocrust and below crust) was a relatively small factor in differentiating the metatranscriptomes, suggesting that the metabolically active bacteria were similar between shallow soil horizons. The dry samples were consistently enriched for hydrogenase genes, indicating that molecular hydrogen may serve as an energy source for the desiccated soil communities. The water pulse was associated with a restructuring of the metatranscriptome, particularly for the biocrusts. Biocrusts increased transcripts for photosynthesis and carbon fixation, suggesting a rapid resuscitation upon wetting. In contrast, the trampled surface soils showed a much smaller response to wetting, indicating that trampling altered the metabolic response of the community. Finally, several biogeochemical cycling genes in carbon and nitrogen cycling were assessed for their change in abundance due to wetting in the biocrusts. Different transcripts encoding the same gene product did not show a consensus response, with some more abundant in dry or wet biocrusts, highlighting the challenges in relating transcript abundance to biogeochemical cycling rates.\n\nThese observations demonstrate that metatranscriptome sequencing was able to distinguish alterations in the function of arid soil microbial communities at two varying temporal scales, a long-term ecosystems disturbance through foot trampling, and a short term wetting pulse. Thus, community metatranscriptomes have the potential to inform studies on the response and resilience of biocrusts to various environmental perturbations.

microbiology↗

bioRxiv2018

Axenic Aedes aegypti develop without live bacteria, but exhibit delayed development and reduced oviposition.

The mosquito gut microbiome plays an important role in mosquito development and fitness, providing a promising avenue for novel mosquito control strategies. Here we present a method for rearing axenic (bacteria free) Aedes aegypti mosquitoes, which will greatly facilitate mechanistic studies documenting the structure and function of the microbiome. Through feeding sterilized larvae agar plugs containing attenuated Escherichia coli, mosquito development was observed in the absence of living bacteria. Axenic larvae were capable of full development into adults, which laid eggs that were subsequently hatched. However, axenic mosquitoes exhibited delayed development time and reduced egg clutch size in comparison to bacterially colonized mosquitoes. These findings suggest that mosquito development is not dependent on live bacteria, but their phenotype is modulated by the presence of microorganisms. This axenic system offers a new tool in which the mosquito microbiome can be systematically manipulated for a deeper understanding of microbiome host interactions.

microbiology↗